Evaluation of a multiplex PCR assay for concurrent detection of four major mycotoxigenic fungi from foods
Article first published online: 11 JAN 2013
© 2012 Defence Food Research Laboratory © 2012 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 114, Issue 3, pages 819–827, March 2013
How to Cite
Rashmi, R., Ramana, M.V., Shylaja, R., Uppalapati, S.R., Murali, H.S. and Batra, H.V. (2013), Evaluation of a multiplex PCR assay for concurrent detection of four major mycotoxigenic fungi from foods. Journal of Applied Microbiology, 114: 819–827. doi: 10.1111/jam.12100
- Issue published online: 18 FEB 2013
- Article first published online: 11 JAN 2013
- Accepted manuscript online: 7 DEC 2012 08:01AM EST
- Manuscript Accepted: 5 DEC 2012
- Manuscript Revised: 28 NOV 2012
- Manuscript Received: 28 SEP 2012
- internal amplification control;
- multiplex polymerase chain reaction;
- ochratoxin A;
To develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of four major mycotoxin metabolic pathway genes, viz. nor1 (aflatoxin), Tri6 (trichothecene), FUM13 (fumonisin) and otanps (ochratoxin A).
Methods and Results
A mPCR assay with competitive internal amplification control, employing specific primers for each of the aforementioned four genes, was optimized and validated using 10 reference strains and 60 pure culture isolates. The standardized mPCR assay detected all four mycotoxin metabolic genes in artificially contaminated maize samples with a sensitivity of 2 × 103 CFU g−1 for nor1-positive Aspergillus strains, Tri6 and FUM13-positive Fusarium strains and 2 × 104 CFU g−1 for otanps-positive Penicillium strains. When the developed mPCR assay was applied to 40 natural foods, 35% (14 of 40) of the samples were contaminated with either one or more mycotoxins. The mPCR results were further evaluated with high-performance liquid chromatography (HPLC), and in general, both the methods provided unequivocal results.
The current mPCR assay is a rapid and reliable tool for simultaneous specific and sensitive detection of aflatoxigenic Aspergillus strains, trichothecene- and fumonisin-producing Fusarium strains, and ochratoxigenic Penicillium species from naturally contaminated foods.
Significance and Impact of the Study
This mPCR assay could be a supplementary strategy to current conventional mycotoxin analytical techniques such as thin-layer chromatography (TLC), high performance thin layer chromatography, HPLC, etc., and a reliable tool for high-throughput monitoring of major mycotoxin-producing fungi during the processing steps of food and feed commodities.